This proposal outlines four specific aims which focus directly on methodology for use in structure-based approaches to modern drug design. (1) In studies on ligand binding and occupancy in crystals, methods will be developed for estimating the occupancy of a ligand bound in a protein crystal, ad for ascertaining whether solid state ligand binding faithfully reproduces binding in solution. (2) In studies on protein- bound water, methods will be developed to predict sites of tightly-bound water and to extend the information content of structures determined by NMR spectroscopy or x-ray crystallography at medium resolution. (3) In studies on glutathione reductase (GR) and trypanothione reductase (TR), several new inhibitors of GR and TR, based on currently available or newly developed structure-based design methodologies, will be synthesized and evaluated. (4) In studies on extended surface structures known enzyme ligands will be extended to specifically displace bound water molecules. Besides testing these ideas on GR and TR, new lysozyme and HIV protease inhibitors will be synthesized to investigate whether entropy-based incremental improvements in binding affinity can be achieved.